Method for inactivating a virus

ABSTRACT

The present invention concerns a method for inactivating a virus for use in e.g. a vaccine, by treating the virus with an ethyleneimine at a pH of less than 7.0, and methods of treatment with the inactivated virus.

BACKGROUND OF THE INVENTION

The invention relates to a method of preparing viruses for use invaccines.

Inactivation of a virus can alter viral antigens, reducing the safety orefficacy of the vaccine. Ideally, the conditions and agent(s) for viralinactivation would selectively and irreversibly affect the viral genome.

Ethyleneimine monomer (EI) or binary ethyleneimine (BEI) are reagentsused to modify nucleic acids preferentially at N-7, N-3, and N-1 ofpurines and to a lesser extent N-3 of pyrimidines. Alkylating agentsenhance the opening of an imidazole ring of N-7 alkylated purines (e.g.,guanine), thereby arresting replication. EI alkylates guanosine to formN-7 (aminoethyl)guanosine which has a higher imidazole ring opening ratethan does N-7 (alkylguanosine). EI also modifies non-genomic componentsof the viron or nonviral biomolecules.

One undesirable side-effect of this nonspecific reactivity is thedisruption of viral particles which reduces immunogenicity. Chemicalmodification of even a single amino acid can significantly change theresistance of protein toward proteinases and may reduce stability ofvaccine during storage due to proteolysis of modified viral proteins.Preferential inactivation of protective epitopes can contribute to animbalanced immune response and to potentiation of disease duringsubsequent infection. Modification of capsid components can inhibit theintracellular processing of viral proteins which are necessary forpresentation of epitopes to T-cells. Third, modification of amino acidresidues of viral proteins reduces viral antigenicity. Finally, chemicalmodification of proteins present in the initial virus-containing mattermay alter their antigenic specificity. This is the primary cause ofallergic reactions in humans after booster doses of inactivated rabiesvaccine. Despite their inherently low selectivity, ethyleneimine monomer(EI or BEI) have been used as agents for production of the killedantiviral vaccines.

SUMMARY OF THE INVENTION

The invention features a method for inactivating a virus, which methodincludes treating the virus with an inactivating amount of a compositionincluding an ethyleneimine at a pH less than 7.0 (e.g., a pH between 5.5and 7.0, or a pH less than 6.8). Preferably, the immunogenicity of thetreated virus is enhanced compared to the immungenicity of the samevirus treated at a pH greater than or equal to pH 7.0 (e.g., pH 7.5,8.0, or preferably 7.0). Immunogenicity and inactivation can be measuredby methods known in the art, including but not limited to those methodsdescribed herein. An ethyleneimine is selected from monomeric andoligomeric forms of ethyleneimine. The concentration of an ethyleneimineis determined by weight/volume (w/v) (e.g, between 0.01% and 1.0% w/v,or between 0.01% and 0.5% w/v). Mixtures of monomeric and oligomericethyleneimines, or mixtures of oligomeric ethyleneimines, can be used.Oligomeric includes between 2 and 8 units, e.g., dimeric, trimeric,branched or straight tetrameric, and so on. Examples of viruses includepolio, rabies, yellow fever, Japanese encephalitis, tick-borneencephalitis, measles, mumps, Ross River virus, rotavirus, and rubella.

The invention also features a method for inactivating a virus, whichmethod includes contacting the virus with a composition includingmonomeric ethyleneimine at a pH less than 7.0. The pH will have beendetermined by a method which includes (a) treating a plurality ofsamples of the virus with an inactivating amount of a compositioncontaining monomeric ethyleneimine at a plurality of different pHvalues; (b) measuring in each treated virus sample a characteristicselected from viral inactivation and immunogenicity; and (c) selectingthe pH which provides, relative to the other pH values of step (a), ahigher viral inactivation or a higher immunogenicity, or a combinationthereof.

Other features and advantages of the invention will be apparent from thefollowing description and from the claims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention features a method of inactivating a virus at a pH lessthan 7.0 by the action of an electrophilic inactivating agent, e.g., anethyleneimine (EI). Treatment at an acidic pH inactivates viral nucleicacids with surprisingly less adverse reaction of an electrophilicreagent with viral proteins, when compared with treatment at a pH of 7.0or higher. Adverse reactions with viral proteins can lead to decreasedresistance to proteinases, distintegration of viral particles,alteration of viral antigens, and inhibition of intracellular processingof viral proteins. A virus treated by the disclosed method is thereforelikely to be more stable during storage and to have betterimmunogenicity and antigenic specificity.

Within the pH interval 6.5-8.5, nucleic acid bases remain uncharged. Inturn, the nucleophilicity and the rate of modification withelectrophilic reagents remain substantially the same. However,alteration of pH affects the nucleophilicity of the amino acid residueshaving protonizable heteroatoms. For instance, protonation of theimidazole ring in histidine and tryptophan or the hydroxy group oftyrosine almost completely prevents the reaction of the amino acid withan electrophilic reagent.

Other factors make it difficult to predict the effects of a lowered pHon nonspecific reactivity of proteins with EI. Existence ofintramolecular interactions among viral proteins and between viralproteins and other components of the medium can significantly alter bothaccessibility and pKa of these amino acids. Alteration of inactivationconditions may lead to alteration in the higher structure of viralcomponents (both proteins and nucleic acid) and, in turn, affect therate of the component reactions with EI.

Optimization of inactivation (e.g., minimum time interval for treatment,concentration of EI, ionic strength, temperature, and pH) would decreasethe extent of side reactions affecting immunogenic potency andspecificity of killed vaccines. Optimal conditions for inactivatingviruses for the purpose of preparing a killed viral vaccine include thefollowing: (1) determination of the virus infectivity inactivation rateconstants as a function of pH between 6.5 and 8.0 (or between 5.5 and7.0, or between 5.0 and 7.0); (2) determination of the selectivity ofthe virus infectivity inactivation (extent of modification of virionproteins and nonviral proteins) at different pH values; and (3)determination of the potency of vaccines inactivated at different pHvalues.

OTHER EMBODIMENTS

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, other embodiments are also within the claims.

Without further elaboration, it is believed that one skilled in the artcan, based on the description herein, utilize the present invention toits fullest extent. The following specific examples are, therefore, tobe construed merely as illustrative, and not limitative of the remainderof the disclosure in any way whatsoever. Publications mentioned hereinare hereby incorporated by reference.

EXAMPLE 1

Determination of the virus infectivity inactivation rate constants as afunction of pH between 6.5 and 8.0

The virus inactivation rate constant is used to determine theapproximate inactivation conditions required to produce a safe vaccine,as a function of the starting titer of live virus and the extent ofinactivation required. Inactivation should generally proceed for a timecalculated to give an adequately low titer of viable virus to preclude ameaningful incidence of live virus infection. For many viruses, 12 to 20logs of inactivation results in a vaccine calculated to deliver lessthan a single viable virion for every one million vaccine recipients.Indirect methods, such as PCR, used to determine of the extent of virusinactivation by other inactivating agents are misleading, althoughdirect determination of viral infectivity cannot be performed.

Determination of the inactivation rate constant is performed as follows:Freshly distilled ethyleneimine (EI) is dissolved in 0.1M NaCl and 0.1MMOPS buffer or 0.1M Tris HCl buffer for a final concentration of 0.5%(0.125M), 0.1% (0.025M) and 0.02% (0.005M) of EI. The final pH should be6.5, 7.0, 7.5 or 8.0, respectively. The reagent solution is incubated at20° C. or 37° C. and added to purified virus suspension (initial titer˜10⁵ -10⁸ PFU./ml) preincubated in the same buffer, at the same pH, andat the same temperature. Aliquots after incubation of the reactionmixture for 0, 1.0, 2.0, 4.0, 8.0, and 16 hours are removed, the excessof the reagent quenched immediately by addition of 0.1 volume of 1.0Mthiosulfate (neutralized to the same pH), incubated at the sametemperature for 0.5-1.0 hours, then cooled to 0° C. and kept at thistemperature before further dilution with cultural medium and plating.Control--the same reaction mixture without EI. Following inactivation,the virus is titered in the standard fashion used to determine theconcentration of replication competent virions remaining. The rateconstant of inactivation (k, h-1, mM-1) is calculated as: ##EQU1##where: A₃ final concentration of the reagent in the reaction mixture inmM; t₃ time of incubation with the reagent in hours; S_(o) and S_(t3)titer of the virus before and after t hours of inactivation.

The pH must be precisely measured (±0.05) and kept constant before andduring inactivation. The temperature of the reaction mixture must beconstant (±1.0° C.). The virus should be preincubated at the giventemperature as long as it is necessary to observe the expectedexponential (semilogarithmic) kinetics of inactivation. Neither reagentsolution nor virus suspension must contain determinable (>1 mM) amountsof phosphate, citrate and other sources of oligoanions. Virus suspensionmust not contain natural or artificial oligocations (spermine,spermidine, etc.) or other compounds able to change the higher structureof the viral genome or nucleoprotein. Genome lesion repair,recombination of genome at high multiplicity of infection, orreassortment of viral genome containing fragmented genome may lead tonon-exponential inactivation kinetics. This may be suspected on thebasis of knowledge of the viral genome. It can be observedexperimentally and taken into account in further studies.

EXAMPLE 2

Determination of the selectivity of the virus infectivity inactivationat different pH's with regard to virion proteins and nonviral proteins

Selectivity is the ratio of the viral genome modification to the rate ofmodification of other components of the reaction mixture (viral or otherproteins, glycoproteins, polysaccharides, etc.). Modification of asingle nucleoside residue in the viral genome prevent its completereplication and, hence, reproduction of the virus. On average,appearance of 2.3 modified residues (preventing complete genomereplication) per genome leads to a one log decrease in virusinfectivity.

The most important adverse reactions may be caused by degradation ofvirion or by modification of other than genome biopolymers: proteins,glycoproteins and polysaccharides. Degradation of the virion leads tosignificant (several orders of magnitude) decrease in the immunogenicitythat requires proportional increase in the amount of vaccine necessaryto elicit the proper immune response. The other and more seriousconsequences are determined by modification of other (besides genome)components of the reaction mixture, mainly of viral and, in many cases,of other proteins and macromolecules present in the reaction mixture.

Fortunately, each genome contain much more exposed reactive residues(purines in nucleic acids are the main target for electrophilicreagents) than any other molecule. Moreover, only genome is, as a rule,in only a single copy per virion. All other molecules that are presentedby many copies (excessive copies of each macromolecule per virion), and,therefore, their modification become visible only when many copies weremodified in the same manner during infectivity inactivation. Moreover,the reactivity of nucleic bases in the pH range 6-8 remain essentiallythe same, while the reactivity of amino acid residues in proteins ischanged dramatically in this pH interval and is much lower when beingprotonated. Hence, the maximum specificity of the genome modificationmay be obtained by the lowest possible pH that, in the case of EI, ispossible at pH about 6.5. In the case of EI (pK_(a) ˜8.1) this may leadto a small increase in the rate of inactivation, but can decreasesignificantly the amount of reactive (non-protonated) amino acidresidues, and, hence, decrease the rate of their modification relativeto inactivation.

Several methods may be used to determine the extent of proteinmodification, including isoelectric focusing and immunologicalmeasurements. In either case, a standard preparation of virus is dividedand inactivated at pH's 6.5, 7.0, 7.5, and 8.0 at the appropriatetemperature and time to achieve the desired extent of inactivation ateach pH. In each case, control preparations of virus are treatedidentically except for the omission of EI. Following inactivation andquenching with thiosulfate as outlined above, portions of theinactivated and control virus preparations are subjected to isoelectricfocusing using standard techniques. Since modification of amino acidresidues with EI leads to an increase in the pK_(a) of the protein,isoelectrofocusing is a sensitive method for detecting proteinmodification. Protein bands can be detected in the IEF gels by anystandard technique such as Coomassie blue staining, silver staining orautoradiography if labelled virus has been used. The results will revealvariability in the extent of alteration of the banding pattern among thedifferent conditions of inactivation, and will allow the choice of thecondition resulting in least modification of proteins. If the virus issuspended in protein-containing media during inactivation, modificationof the medium protein should also be determined by IEF following removalof the virus from the inactivation mixture by high speed centrifugation.

As an additional test of selectivity of inactivation, the inactivatedvirus and control preparations may be subjected to immunologicalassessment by ELISA or a similar standard assay, designed to determinethe reactivity of virion proteins with standardized immunologicalreagents such as a panel of monoclonal antibodies. To perform thisassessment, the inactivated and control virus preparations prepared atdifferent pH's are subject to ELISA under defined conditions. Reductionsin antigenicity will usually be found under some conditions ofinactivation. Under most conditions, these conditions should be similar(and preferably the same) as those determined by IEF to result inenhanced modification of proteins. In some cases, detectablemodification of proteins may be found only under conditions leading toinactivation of the virus infectivity (theoretical value) by more than50 logs. Even in this case mainly a single hit reaction(s) with proteins(monomodified proteins) can be detected, although modification of thevirus genome is as high as several hundred nucleoside residues perpolynucleotide molecule.

EXAMPLE 3

Determination of the potency of vaccines inactivated at different pH's

Following determination of the extent of modification of proteins, theinactivated virus preparations are tested for vaccine potency. Toaccomplish this, the virus preparations inactivated at various pH's areadministered to the appropriate animal under conditions designed toprovide an effective immune response, e.g., three to fouradministrations with or without an immunological adjuvant at suitableintervals. Ordinarily, each vaccine preparation will be given at variousdose levels to allow determination of a dose response relationship, ameasure of potency. At baseline and one week following eachadministration of vaccine, blood or other fluid is tested for antibodyagainst viral components. Following the final vaccine administration,the animal is tested for protective efficacy by administration ofwild-type virus and observation for consequences of viral infection. Inthis way, potency of the vaccine produced under each condition can beevaluated. For some viruses, assessment of either immune response orprotective efficacy may be the more valuable parameter for prediction ofvaccine effectiveness in humans.

What is claimed is:
 1. A method for inactivating a virus selected fromthe group consisting of polio virus, rabies virus, yellow fever virus,Japanese encephalitis virus, tick-borne encephalitis virus, measlesvirus, mumps virus, Ross River virus, rotavirus, and rubella, saidmethod comprising treating said virus with an inactivating amount of acomposition comprising an ethyleneimine at a pH less than 7.0.
 2. Themethod of claim 1, wherein said pH is between 5.5 and 7.0.
 3. The methodof claim 1, wherein said pH is less than 6.8.
 4. The method of claim 1,wherein the concentration of said ethyleneimine in said composition isbetween 0.01% and 0.5 % w/v.
 5. The method of claim 1, wherein saidethyleneimine is monomeric.
 6. The method of claim 1, wherein saidethyleneimine is oligomeric.
 7. The method of claim 1, wherein theimmunogenicity of the treated virus is enhanced compared to theimmunogenicity of the same virus treated at pH 7.0.
 8. A method forinactivating a virus selected from the group consisting of poliovirus,rabies virus, yellow fever virus, Japanese encephalitis virus,tick-borne encephalitis virus, measles virus, mumps virus, Ross Rivervirus, rotavirus, and rubella, said method comprising contacting saidvirus with monomeric ethyleneimine at a pH less than 7.0 which has beendetermined by(a) treating a plurality of samples of said virus with aninactivating amount of monomeric ethyleneimine at a plurality ofdifferent pH values; (b) measuring in each treated virus sample acharacteristic selected from viral inactivation and immunogenicity; and(c) selecting the pH which provides, relative to the other pH values ofstep (a), a higher viral inactivation or a higher immunogenicity, or acombination thereof.